The purpose of these studies is to establish a better understanding of the energy metabolism in tissues, in vivo. Towards this goal, the laboratory concentrates on the use of non-invasive and non-destructive techniques to evaluate the biochemical and physiological function of the heart and skeletal muscle with regard to energy metabolism. The following major findings were made over the last year: 1). We continued our efforts in exploring the use of NADH-enzyme dependent fluorescence recovery after photobleaching (NADH-EDFRAP). NADH is the primary energy source for most cellular functions. NADH EDFRAP potentially provides a method of directly monitoring the turnover of this critical molecule within intact cells. Towards a better understanding of the use of the NADH photolysis reaction in the evaluation of cellular energy metabolism, the photochemistry of the NADH photolysis reaction was evaluated using NMR, mass spectroscopy and classical enzymatic techniques. These studies revealed that the photolysis of NADH is primarily an oxidation reaction generating NAD+, or the normal metabolic substrate for NADH generation in the cell, along with 2 free radical molecules/NADH. Using this information, kinetic models for the use of NADH-EDFRAP in isolated enzyme systems and mitochondria were developed and tested. The isolated mitochondria studies revealed that the generation of NADH is far from equilibrium, as previously believed, and significantly contributes to the overall rate limitation of oxidative phosphorylation. This unique approach of perturbing metabolic processes in intact tissues should provide valuable new information on the regulation of numerous metabolic processes in the intact cell. 2) The binding of NADH in the mitochondria matrix was evaluated using fluorescence lifetime and emission spectroscopy. These studies revealed a high affinity site (Kd 20 uM) for NADH binding with a capacity of near 50% of the total NADH in the matrix. These high affinity binding sites are not related to the oxidation of NADH by oxidative phosphorylation based on the kinetics of NADH oxidation. These results have numerous implications in evaluating the chemical activity of NADH in the mitochondrial matrix as well as the detection of NADH using various imaging techniques. 3) A mathematical network model is being developed to create a ?consensus? model of mitochondrial function and oxidative phosphorylation. Using this approach we have been able to combine the results from numerous laboratories around the world into a working hypothesis of mitochondrial function that is useful in guiding hypothesis building and experimental design. 4) Minimally invasive, multi-photon microscopy in intact animals is being utilized to begin evaluating sub-cellular metabolic processes within cells under normal in vivo conditions. Initial studies in the intact mouse skeletal muscle have shown that following intercellular regional differences in mitochondria function and plasma membrane potentials can be observed, in vivo. In addition, the optical absorption properties of several tissues were evaluated to establish the optimal wavelengths and stratagies for multiphoton exciation schemes. This optical microscopy approach provides an important link between classical cell biology and whole animal physiology studies permitting the evaluation of cellular events in the intact animal.